The most abundant intracellular divalent cation, intracellular Mg concentration is tightly controlled. Work from this and other laboratories suggests Mg2+ plays a fundamental regulatory role in cellular metabolism and growth. Thus, all cells likely possess interesting mechanisms for sensing Mg2+ and for controlling its passage through the cell membrane. Moreover, Mg2+ is unique among biological cations in possessing the largest hydrated radius and the smallest atomic radius. As a consequence, in an aqueous environment, its volume change from atomic to hydrated cation is almost 400-fold, 20 times larger than that of any other common biological cation. Since it is the atomic cation that traverses the bilayer, the unique chemical properties of aqueous Mg2+ may be mirrored by unique molecular properties of Mg2+ transporters. The CorA transporter of Salmonella typhimurium represents a new family of transporters lacking significant similarity to other known proteins, yet widespread among Gram-negative bacteria. While unambiguously an integral membrane protein, 29% of CorA's 316 amino acids bear frank charge. The membrane topology of CorA shows that the protein consists of two essentially independent domains: an N-terminal periplasmic domain of about 230 amino acids and an 80 amino acid C-terminal domain comprised of only three transmembrane segments. Further, we have recently cloned a second new and again, highly unusual class of Mg2+ transporter present in both Gram-negative and Gram-positive bacteria. Aim I will use both molecular and chemical crosslinking methods to determine if CorA functions as a homo-oligomer. Aims II and III treat the periplasmic and membrane domains of CorA as independent structures. A model of the membrane domain will be constructed from cysteine substitution and crosslinking experiments and from site-directed mutagenesis studies to determine residues important for cation binding. The structure of the periplasmic domain will be investigated by genetic selection for mutants and by limited site-directed mutagenesis. The domain will be purified for preliminary NMR and crystallization studies. Together, data in Aims II and III will enable construction of a complete three-dimensional model of the unique CorA Mg2+ transport protein. Aim IV will identify further new classes of Mg2+ transporters, characterize their properties and determine their phylogenetic distribution. Overall this project should lead to a greater understanding of the basis of Mg2+ transport and cellular Mg2+ homeostasis.